Calcium-activated neutral protease

D. E. Croall, G. N. DeMartino, Calcium Activated Neutral Protease (Calpain) System Structure, Function and Regulation , Physiol. Rev. 1991, 71, 813-847. 

Reddy, P.A., Anandavalli, T.E., and Anandaraj, M.P., 1986, Calcium activated neutral proteases (milli-and micro-CANP) and endogenous CANP inhibitor of muscle in Duchenne muscular dystrophy (DMD), Clin Chim Acta, 160, pp 281-288. 

Calcium ion is a known activator of many biochemical processes. The calcium-activated neutral protease (CANP) plays an important role in postmortem tenderizing of meat. The function of the metal ion in such an enzyme is believed to be either neutralization of the charges on the surface, by preventing electrostatic repulsion of subunits, or effecting of a conformational change required for association of the subunits. Thus the metal ions must be present in a specific state to perform this function. 

Calcium activated neutral protease calcium ATPase in glucose-6-phosphate dehydrogenase deficiency hemizygotes. 

Belcastro, A.N., Skeletal muscle calcium-activated neutral protease (calpain) with exercise, J Appl Physiol, 74, 1381, 1993. 

Activation of Calpain (Calcium-Dependent Neutral Protease).147... 

Thermolysin (EC 3.4.24.4) a heat-stable, zinc- and calcium-containing neutral protease, M, 37,500, from Bacillus thermoproteolyticus, with a substrate specificity similar to that of Subtilisin (see). After one hour at 80 °C, T. still has 50% original activity. This high heat stability of T. is attributed to the large number of hydrophobic regions and the presence of four bound calcium ions, which serve in place of disulfide bridges (T. contains no disulfide bridges) to maintain the compact shape of the molecule. T. is neither a thiol nor a serine enzyme. 

Takai, Y., Yamamoto, M., Inoue, M., Kishimoto, A., and Nishizuka, Y., 1977, A proenzyme of cyclic nucleotide-independent protein kinase and its activation by calcium-dependent neutral protease from rat liver, Biochem. Biophys. Res. Commun. 77 542. 

A few enzymes, such as the previously mentioned CNP, are believed to be fairly specific for myelin/oligodendro-cytes. There is much more in the CNS than in peripheral nerve, suggesting some function more specialized to the CNS. In addition, a unique pH 7.2 cholesterol ester hydrolase is also enriched in myelin. On the other hand, there are many enzymes that are not myelin-specific but appear to be intrinsic to myelin and not contaminants. These include cAMP-stimulated kinase, calcium/calmodulin-dependent kinase, protein kinase C, a neutral protease activity and phosphoprotein phosphatases. The protein kinase C and phosphatase activities are presumed to be responsible for the rapid turnover of MBP phosphate groups, and the PLP acylation enzyme activity is also intrinsic to myelin. 

The Ca2+-dependent neutral proteases called calpains are found within the cells of higher animals. The 705-residue multidomain peptide chain of a chicken calpain contains a papain-like domain as well as a calmodulin-like domain.328 It presumably arose from fusion of the genes of these proteins. At least six calpains with similar properties are known.329 Some have a preference for myofibrillar proteins or neurofilaments.330 They presumably function in normal turnover of these proteins and may play a role in numerous calcium-activated cellular processes.331-3323... 

Proteases contribute to the inflammatory response to injury, forming a final common pathway that leads to BBB breakdown, hemorrhage, and cell death. After traumatic and ischemic injuries, there is a buildup of lactate, which is increased with hyperglycemia. Acidosis leads to release of acid hydrolases, which are destructive enzymes that attack cellular components, including membranes, resulting in cell necrosis. In situations where the pH remains neutral, increases in intracellular calcium and cytokines cause induction of neutral proteases. The main neutral proteases are the extracellular matrix-degrading MMPs, plasminogen activator/plasmin, and caspases. 

Fig. 4.7.S. Effect of pH on the adsorption of neutral protease on Sepharose 4B substituted with triethylenetetramine, succinic anhydride, triethylenetetramine and chloroacetyl-D-phenylalanine. Crude enzyme (10 mg) was dissolved in 0.1 ml of equilibrating buffer and applied to an affinity column (220x6 mm) equilibrated with 1(X) mM sodium chloride, containing 5 mM Tris (pH 7.5 or 7.0) or 5 mM 2-( -morpholino)ethanesulfonic acid (pH 6.5 or 6.0). After elution for 1 h at 25 ml/h, each column was washed with 100 mM sodium chloride, 10 mM calcium chloride, 50 mM Tris (pH 9.0), as indicated by the arrows. Neutral protease and subtilisin are identified by catalytic activities toward 3-(2-furylacryloyl)-glycyt-L-leucinamide (FAGLA) and acetyl-JV-tyrosine ethyl ester (ATEE), Reproduced with permission from Ref. 62.

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